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Frequently Asked Questions

Please use the information below to troubleshoot any common problems you may have. If you cannot find your question here or the answer is not sufficient then please contact us immediately.

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Transportation of the V2a Kit™ - Vasculogenesis to Angiogenesis:

Q. Is V2a Kit™ adversely affected by transport to our lab?

A. V2a Kit™ is not a live kit and is extremely robust during transportation.

Receiving V2a Kit™ - From Vasculogenesis to Angiogenesis:

Q. What should I do when the kit arrive at our laboratory?

A. V2a Kit™ must be unpacked immediately upon arrival. It is important that the cells are transferred directly from the dry ice to liquid nitrogen. Ensure that all components are stored at the correct temperature according to the V2a Kit™ Protocol.

Q. Following the first medium change, cells detach with areas becoming devoid of cells. Damage may be particularly evident at the side of wells, and some floating dead cells may be seen. What causes this?

A. This is caused by addition of optimised medium too quickly. The problem may occur through the use of a single channel automatic pipette, rather than a serological pipette as recommended. Wells may recover, but this depends on the extent of the damage and wells will not react in the same way across the complete plate.

Q. What is the typical expiry date for V2a Kit™?

A. V2a Kit™ has a typical shelf life of 3-6 months. Seeding and growth supplements are supplied frozen to be added to the medium when required.

Use of V2a Kit™ - From Vasculogenesis to Angiogenesis:

Q. How should test articles be prepared for application to V2a Kit™?

A. Preparation of test articles needs to be conducted bearing in mind that V2a Kit™ is extremely sensitive to pro- and anti-angiogenic factors. Replacement of the optimized growth medium with significant amounts of buffer or other culture medium and addition of solvents can themselves have a direct effect on the formation of tubules as they would in vivo. Current recommendations for solvent use are as follows: Ethanol, dimethylformamide and DMSO can be tolerated up to 0.1%. There is a requirement for at least 80% optimized growth medium (see the kit protocol for more information).

Q. Once the frozen cells have been thawed and seeded for the angiogenesis assay. How confluent should they be?
V2a D2 QC140918 well D4

A. Please see the photograph, taken on day 2 (section 8.2.5 of the V2a protocol ), ie the day after the cells were seeded.

Q. What is the Optimal Day for Staining V2a Kit Vasculogenesis to Angiogenesis Kit, plates?

A. A series of in house experiments conducted to determine the optimal day for staining revealed that this is day 14. The V2a protocol specifies that the assay is ended and stained on day 14. Observations from the experiments revealed that:

  • there was no correlation between staining day and the quality of the staining
  • differences in responses between suramin, untreated and VEGF could be seen as early as day 7
  • staining late (day 16-18) provided a greater difference between suramin and untreated controls, and a greater overall level of tubule development. However, there was a greater incidence of cell sheet disruption with staining days 16 - 18.
Q. Is it possible to leave V2a Kit Angiogenesis Cultures growing over weekends without a media change?

A. Yes. In house experiments conducted to determine whether 3 day media changes over weekends have a detrimental effect on the performance of a V2a kit compared to 2 day media changes showed that there was no marked effect between a 2 or 3 day media change. All cultures reached confluence after 6 days of culture. In addition, there were no observable trends indicating that 3 day media changes had any detrimental effect on tubule length and the number of branch points for the V2a kit.

Q. What is the Optimal Day to add Pro- and Anti-Angiogenic Factors to a V2a kit Vasculogenesis to Angiogenesis?

A. A series of In house experiments conducted to determine the optimal day for adding the Pro- and Anti-Angiogenic Factors factors VEGF and suramin respectively on days 1, 2, 3, 4, 5, and 6 and their effects were compared.

Results showed that adding VEGF and suramin on day 3 produced the largest difference in the number of junctions (see figure 1). The experiments also showed that adding VEGF and suramin on day 2 produced the largest difference in the number of tubules and total tubule length (see figures 2 and 3).

This suggests that VEGF and suramin should be added at day 2 or 3 in order to demonstrate the biggest effect on angiogenesis.

Figure 1 shows the average number of junctions formed as a % difference to untreated wells, for wells treated with VEGF and suramin wells on days 1, 2, 3, 4, 5 and 6.

Figure 1

Figure 2 shows the average number of tubules formed as a % difference to untreated wells, for wells treated with VEGF and suramin on days 1, 2, 3, 4, 5 and 6.

Figure 2

Figure 3 shows the total tubule length as a % difference to untreated wells, for wells treated with VEGF and suramin on days 1, 2, 3, 4, 5 and 6.

Figure 3
Q. No tubule formation occurred. Why?

A. There are many possible reasons for this, including the following:

  1. High concentrations of angiogenic inhibitors have prevented tubule formation. Check control wells.
  2. The solvent used to dissolve test compounds may be at a toxic concentration. Run one or more solvent control wells.
  3. Cultures have died. This may be due to a number of reasons, including:
    1. Cells were allowed to become dry between medium changes. Aspirate old medium from only a few wells at a time and replace with fresh optimized medium before aspirating the next wells.
    2. Final concentration of solvent, in which treatment is dissolved, is too high. Make a more concentrated stock solution of the test compound and dilute this further in medium.
    3. Ensure all incubations are at 37°C and in a 5% CO2 humidified atmosphere.
Q. What has caused the cell sheet to detach from the well surface?

A. Cultures have been left to grow for too long. Try to fix and stain cultures before this occurs. Contact Cellworks if this problem is encountered before the end of the protocol. If the sheet is still partially attached to the well, attempt to gently fix and stain the tubules.

Q. Why are cells floating in the well?

A. There are two possible reasons for this:

  1. This can be the result of adding fresh medium too quickly. Always add fresh medium very gently to prevent dislodging cells. Never use micropipettes for adding medium.
  2. Cultures have died. See above for possible reasons.
Q. A small number of wells are contaminated. What can be done?

A. It is important to act promptly to contain the contamination. Use your laboratory's preferred method for this, or refer to Appendix I in the V2a Kit™ protocol.

Staining V2a Kit™ - From Vasculogenesis to Angiogenesis:

Q. No tubule staining can be observed. Why?

A. Check that antibodies were added in the correct order.

Q. What causes high background staining?

A. High background staining may occur following overlong exposure to the final staining solution (substrate). To address this problem, colour development may be monitored under a light microscope. The development of a permanent stain only takes a few minutes.

Q. What causes faint staining of the tubules?

A. There are many possible reasons for this, including the following:

  1. Antibodies were not added at the correct concentration.
  2. Antibody incubation was too short or was performed at the incorrect temperature.

Cultures were left to grow for too long. The matrix is preventing adequate penetration of the primary antibody. Leave the primary antibody to incubate at 6°C overnight and the secondary antibody for 8 hours at 6°C before addition of substrate as usual.

Staining solutions may have been made up too early. These solutions are light sensitive and must be made up immediately prior to use.

Build a Bespoke Angiogenesis Assay:

Q. Do you have a protocol for a bespoke angiogenesis assay using your cells and media?

A. Yes, please click here to view the protocol

Image Capture:

Q. When capturing images, what area of the well should be photographed?

A. During the V2a assay, you may find that HUVECs adhere in greater numbers around the edge of the wells. This is one of the main difficulties in quantifying the results of the V2a assay as the decisions regarding what regions to photograph can be very subjective. In response to this, the methodology used at Cellworks is to take up to three images from each well in central standardised positions across all of the wells. This approach may not be suitable for all users. Please contact our Technical Support team if you would like to discuss a methodology to suit your particular assay.

Q. What magnification should I use for capturing images for analysis in Angiosys 2.0?

A. Cellworks have found a total image magnification of 8x is suited to analysis using the Angiosys 2.0. Total image magnification can be calculated by multiplying the objective lens magnification by the magnification of the adapter used for your image capture equipment.

Higher magnifications can lead to a ladder-type effect when skeletonising the image and lower magnifications can lead to spurious joining of tubules during dilation operations.

Q. What image formats can be used with Angiosys 2.0?

A. AngioSys will accept images in the following formats:

  • TIFF
  • BMP
  • GIF
  • JPEG
  • PNG
Q. What size of image can be used with Angiosys 2.0?

A. Angiosys 2.0 should be able to process any size of image in the correct format, however the analysis process proceeds pixel by pixel so larger images will require more processor power and more time to complete. Cellworks recommend that images are resized to 1200 pixels x 800 pixels for a balance of image clarity and processing time.

Complaints:

Any complaints about the kit performance are dealt with swiftly, and fully investigated. Please contact us.

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